Mitochondria are tiny organelles found within virtually all of our body's cells. They control a number of important cellular processes, including energy production, fat metabolism, steroid synthesis, and programmed cell death. Recently, multiple studies have shown that inherited or acquired mitochondrial dysfunction can give rise to a host of rare metabolic disorders, as well as many common diseases, including type 2 diabetes mellitus, heart failure, neurodegeneration, and the aging process itself. Mitochondria are complex structures, consisting of an estimated 1500 proteins. The majority of these proteins are encoded in the cell's nucleus, produced in the cytosol, and then imported into the mitochondrion. At present, we only know about 750 of the estimated 1500 mitochondrial proteins. Because mitochondria contribute to so many rare and common human diseases, it's important that we systematically identify all the proteins that constitute this organelle and begin to understand how they function together in health and in disease. Availability of complete mammalian genome sequences, in combination with new protein detection and microscopy technologies, now provide a special opportunity to systematically and comprehensively identify all the protein components of mammalian mitochondria, as well as how they function together. In the current application, we propose to use a multidisciplinary approach that blends protein biochemistry, computational genomics, and imaging, to construct a protein parts list for mammalian mitochondria. This project will provide an important foundation for systematic approaches to mitochondrial function, which will be extremely important in the coming years as we link its activity to common human diseases, such as type 2 diabetes mellitus. Moreover, the protein catalog that we generate will immediately provide a rich source of candidate disease genes for rare mitochondrial disorders.